This invention relates to an improved type of rapid scan spectrophotometer and method of using it in several modes to obtain the absorption characteristics of clear low molecular weight solutions. More particularly, the spectrophotometer of this invention utilizes a technique for accurately controlling the spectrum being observed at a known time and subsequently processing the transmitted spectrum to obtain the absorption characteristic of the material being observed.
Although electronic absorption (uv-visible) spectrophotometry is an attractive investigative tool for clear, low molecular weight solutions, the application of this technique to the study of biological preparations is not straightforward, primarily because these complex and heterogenous systems show broad overlapping absorption bands which scatter light strongly. Where the parameter of interest is not the total absorbance, but the change in absorbance, several prior art spectrometers have been found useful. In the prior art split-beam spectrometer light from a monochromator is alternately passed through two identical samples which are subjected to different conditions. The difference in absorption between the samples is then recorded as a function of wavelength. This approach allows the measurement of small absorbance changes on a large background. However, if the amount of scattered light is time dependent, or if it changes as a consequence of the change in experimental conditions, false absorbance changes will be recorded.
The prior art dual wavelength spectrometer provides a means of correcting for these scattering changes. As the names implies, light of two different wavelengths is used, but both beams pass through the same sample. Variations in light intensity at some reference wavelength at which there is no absorbance change will reflect only scattering effects. To the extent that the relative changes in scattered light are the same at two neighboring wavelengths, the intensity at the reference wavelength provides a continuous correction for scattering occurring at neighboring wavelengths. Also, because the dual wavelength spectrometer rapidly monitors light intensities at a specified pair of wavelengths, it can be used for kinetic studies.
It is therefore a primary object of this invention to provide a rapid-scan spectrophotometer which can operate in a variety of spectroscopic modes, two of which are analogous to those just discussed. The controlled-scan spectrophotometer (CSS) of this invention has been operated as a dual-wavelength spectrophotometer, but one which uses only a single monochromator.
The CSS has also been used to obtain the complete absorbance spectrum, corrected for light scattering changes in the same way that the dual-wavelength approach provides correction at a single wavelength. This latter technique has been called "corrected-differential" spectrophotometry.